The Lyme disease spirochete Borrelia burgdorferi induces inflammation and apoptosis in cells from...

JOURNAL OF NEUROINFLAMMATION

Ramesh et al. Journal of Neuroinflammation 2013, 10:88http://www.jneuroinflammation.com/content/10/1/88

RESEARCH Open Access

The Lyme disease spirochete Borrelia burgdorferiinduces inflammation and apoptosis in cells fromdorsal root gangliaGeeta Ramesh1, Lenay Santana-Gould3, Fiona M Inglis2, John D England3 and Mario T Philipp1*

Abstract

Background: Lyme neuroborreliosis (LNB), caused by the spirochete Borrelia burgdorferi, affects both the peripheraland the central nervous systems. Radiculitis or nerve root inflammation, which can cause pain, sensory loss, andweakness, is the most common manifestation of peripheral LNB in humans. We previously reported that rhesusmonkeys infected with B. burgdorferi develop radiculitis as well as inflammation in the dorsal root ganglia (DRG),with elevated levels of neuronal and satellite glial cell apoptosis in the DRG. We hypothesized that B. burgdorferiinduces inflammatory mediators in glial and neuronal cells and that this inflammatory milieu precipitates glial andneuronal apoptosis.

Methods: To model peripheral neuropathy in LNB we incubated normal rhesus DRG tissue explants with liveB. burgdorferi ex vivo and identified immune mediators, producer cells, and verified the presence of B. burgdorferi intissue sections by immunofluorescence staining and confocal microscopy. We also set up primary cultures of DRGcells from normal adult rhesus macaques and incubated the cultures with live B. burgdorferi. Culture supernatantswere subjected to multiplex ELISA to detect immune mediators, while the cells were evaluated for apoptosis by thein situ TUNEL assay. A role for inflammation in mediating apoptosis was assessed by evaluating the abovephenomena in the presence and absence of various concentrations of the anti-inflammatory drug dexamethasone.As Schwann cells ensheath the dorsal roots of the DRG, we evaluated the potential of live B. burgdorferi to induceinflammatory mediators in human Schwann cell (HSC) cultures.

Results: Rhesus DRG tissue explants exposed to live B. burgdorferi showed localization of CCL2 and IL-6 in sensoryneurons, satellite glial cells and Schwann cells while IL-8 was seen in satellite glial cells and Schwann cells. Live B.burgdorferi induced elevated levels of IL-6, IL-8 and CCL2 in HSC and DRG cultures and apoptosis of sensoryneurons. Dexamethasone reduced the levels of immune mediators and neuronal apoptosis in a dose dependentmanner.

Conclusion: In this model, B. burgdorferi induced an inflammatory response and neuronal apoptosis of DRG. Thesepathophysiological processes could contribute to peripheral neuropathy in LNB.

Keywords: Lyme neuroborreliosis, Borrelia burgdorferi, Dorsal root ganglia, Schwann cells, CCL2/MCP-1, IL-6, IL-8,Neuronal apoptosis, Dexamethasone

* Correspondence: [email protected] of Bacteriology and Parasitology, Tulane National Primate ResearchCenter, Covington, LA, USAFull list of author information is available at the end of the article

© 2013 Ramesh et al.; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the CreativeCommons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, andreproduction in any medium, provided the original work is properly cited.

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Ramesh et al. Journal of Neuroinflammation 2013, 10:88 of 14http://www.jneuroinflammation.com/content/10/1/88

BackgroundLyme neuroborreliosis (LNB), the form of Lyme diseasethat affects the nervous system, is manifest in about 15%of patients. Both the central and peripheral nervoussystems may be affected [1-5]. Patients with central ner-vous system (CNS) involvement may complain of head-ache, flu-like symptoms, fatigue, memory loss, learningdisability or depression. Infection of the peripheral ner-vous system (PNS) with Borrelia burgdorferi, the Lymedisease bacterium, may result in facial nerve palsy, pain,sensory loss, or muscle weakness.Clinically, LNB may manifest as meningitis, typically

characterized by lymphocytic pleocytosis in the cerebro-spinal fluid (CSF), meningoradiculitis (also known asBannwarth’s syndrome), cranial neuritis, encephalopathy,peripheral neuropathy and, less commonly, encephalitisand encephalomyelitis. Radiculitis, or inflammation inthe dorsal roots, is the most common manifestation ofuntreated LNB in humans [2,4]. LNB patients may alsoexperience a wide array of neurological symptoms as aresult of white matter inflammation in the brain andspinal cord that results in a sub-acute multiple sclerosis(MS)-like manifestation [6,7].Pathology reports from human cases of LNB have de-

scribed lymphocyte and plasma cell infiltration in themeninges and perivascularly in the nerve roots, dorsalroot ganglia (DRG), as well as demyelination in the brainand spinal cord [8-13]. Typically, peripheral nervous sys-tem Lyme disease is associated with patchy multifocalaxonal loss with epineural perivascular inflammatory in-filtrates or perineuritis [14-18].In an earlier study, histopathological evaluation showed

varying degrees of necrosis in the sensory ganglia of rhesusmacaques that were infected with B. burgdorferi, inaddition to positive immunostaining with monoclonalantibodies against a 7.5 kDa lipoprotein of B. burgdorferi[19]. In an in vivo experiment in which we inoculated B.burgdorferi into the cisterna magna of rhesus macaques,analysis of the CSF within one-week post-inoculationshowed increased levels of IL-6, IL-8, CCL2, and CXCL13,accompanied by a monocytic/lymphocytic pleocytosis.This inflammatory response was concomitant with histo-pathological changes consistent with acute neurologicLyme disease, such as leptomeningitis and radiculitis. Inaddition, we observed elevated levels of neuronal and sat-ellite glial cell apoptosis in the DRG of infected animals ascompared to uninfected controls and documented thepresence of IL-6 in DRG neurons of infected animals [20].The mechanisms underlying the pathogenesis of per-

ipheral LNB are not clearly understood. Based on ourobservations, we hypothesized that B. burgdorferi wasable to induce inflammatory mediators in glial and neur-onal cells and that this inflammatory context precipi-tated glial and neuronal apoptosis. As a model to study

the mechanisms underlying peripheral neuropathy seenin patients with Lyme neuroborreliosis, we obtainedfresh rhesus DRG tissue explants and allowed live Lymedisease bacteria to interact with the tissue explantsex vivo to allow for accumulation of intracytoplasmicproteins. Cryo-sections were stained to detect immunemediators, the phenotypes of producer cells and thepresence of B. burgdorferi spirochetes, and were visual-ized using confocal microscopy. We also set up primarycultures of dorsal root ganglia cells from normal adultrhesus macaques and characterized the cells phenotypic-ally. We then incubated the DRG cultures with live B.burgdorferi. Culture supernatants were used for the de-tection of immune mediators while the cells wereassessed for apoptosis. To determine if inflammationhad a role in apoptosis, the above phenomena were eval-uated in the presence and absence of various concentra-tions of the anti-inflammatory drug dexamethasone.Since DRG also contain Schwann cells, which ensheaththe dorsal roots, we investigated whether B. burgdorferihad the potential to induce inflammation in humanSchwann cells. The results of these experiments are de-scribed below.

MethodsGrowth and preparation of live spirochetesB. burgdorferi B31 clone 5A19 spirochetes, passage 1 to3 were grown to late logarithmic phase under micro-aerophilic conditions in Barbour Stoenner-Kelly (BSK)medium, supplemented with 6% rabbit serum (Sigma, St.Louis, MO, USA) and antibiotics (rifampicin at 45.4 mg/mL, fosfomycin at 193 mg/mL and amphotericin at0.25 mg/mL). Spirochetes were pelleted at 2000 × g for30 minutes at room temperature. At the end of the runthe rotor was left to coast without breaking so as tominimize damage to the live spirochetes. The culturewas washed using sterile phosphate buffered saline (PBS)and resuspended in the working medium at the desireddensity.

Incubation of dorsal root ganglia explant slices with livespirochetesDRG tissue was obtained immediately after euthanasiafrom three normal rhesus macaques and placed in PBSpH 7.2 (Invitrogen, Grand Island, NY, USA) at roomtemperature. The tissue was sliced using sterile number21 scalpels (Personna Medical, Verona, VA, USA). Theslices were placed in separate wells of 12-well plates(Fisher Scientific, Fair Lawn, NJ, USA), each containing2 ml of RPMI 1640 medium (Invitrogen) supplementedwith 10% fetal bovine serum (FBS) (Invitrogen). Live B.burgdorferi spirochetes at a final density of 1 × 107/mLwere added to some wells. Some wells received, inaddition, brefeldin A (Molecular Probes, Eugene, OR,


USA), a fungal metabolite that blocks protein transport[21] at a final concentration of 10 μg/mL. Correspondingcontrol slices were also held in medium plus brefeldin Awithout spirochetes. The DRG slices were then incu-bated at 37°C for four hours in a humidified 5%-CO2 in-cubator. At the end of the four-hour incubation, tissueslices were fixed in 2% paraformaldehyde in PBS pH 7.0(USB, Cleveland, OH, USA) and cryopreserved as de-scribed earlier [22].

Immunofluorescence staining for detection ofintracytoplasmic immune mediatorsFor in situ analysis of intracytoplasmic proteins, frozentissue blocks were cryosectioned into 16-μm sections aspreviously described [22]. A total of ten cryosectionswas evaluated per tissue block from each of the abovethree animals for detection of intracytoplasmic immunemediators. DRG tissue slices were subjected to im-munofluorescence staining as previously described [23].The primary antibodies against various phenotypic mar-kers of cells used were anti-human neurotrophin recep-tor p75 (p75NTR) polyclonal rabbit antibody presentedin rabbit serum used at 1:10 dilution (Millipore, Billerica,MA, USA), anti-human 2′,3′-cyclic nucleotide 3′-phos-phodiesterase (CNPase), clone 11-5B mouse IgG1(Millipore) at 10 μg/mL, anti-human S-100 (Sigma) at1:500, anti-human neuronal protein NeuN, MAB 377clone A60, mouse IgG1 (Millipore) at 1:10, or anti-human glial fibrillary acidic protein (GFAP), at 1:200,clone G-A-5 purified mouse immunoglobulin conjugatedto Cy3 (Sigma). Primary antibodies for immune media-tors were either anti-human IL-6, mouse IgG2a at1:1000 (ProSpec, Ness Ziona, Israel), anti-human CCL2,mouse IgG1 (5 J): SC-32771 at 1:50 (Santa Cruz Biotech-nology, Santa Cruz, CA, USA) or rabbit polyclonal IgGclone ab7814 at 1:50 (AbCam, Cambridge, MA, USA),anti-human IL-8 polyclonal rabbit IgG at 10 μg/mL(RDI, Flanders, NJ, USA). B. burgdorferi was stained witha polyclonal rabbit antibody against whole Borrelia at1:200 (Accurate Chemicals, Westbury, NY, USA) incombination with a Zenon kit Alexa 647 (Invitrogen).Isotype controls (Sigma) at the concentrations of the re-spective primary monoclonal antibodies and universalrabbit negative control (Dako Cytomation, Carpinteria,CA, USA) for rabbit polyclonals were also included. Allprimary antibodies and isotype controls at the appropri-ate concentrations were prepared in PBS containing 10%normal goat serum (NGS, Invitrogen), 0.2% fish skin gel-atin (FSG, Sigma) and 0.02% sodium azide (Sigma), andleft on the slides for one hour at room temperature in ahumidifying chamber. The slides were then washed withPBS-FT buffer (PBS pH 7.4 containing 0.2% FSG and0.02% Triton X-100 (MP Biomedicals, Solon, OH, USA)and then held in this buffer for five minutes followed by a

rinse with PBS-F buffer (phosphate-buffered salinecontaining 0.2% FSG).The relevant secondary antibodies, goat anti-rabbit con-

jugated to one of the Alexa fluorochromes, Alexa 488(green), 568 (red) or 633 (blue) (Invitrogen), at a dilutionof 1:1000 in PBS containing 10% NGS, 0.2% FSG and0.02% sodium azide were applied to the tissues and left inthe humidified dark slide chamber at room temperaturefor 30 to 45 minutes. In some cases the Zenon Rabbit IgGlabeling kit was used (Invitrogen). Slides were washed withPBS-FT buffer and rinsed with PBS-F buffer as describedabove and then mounted in anti-quenching medium(Sigma). The stained and mounted slides were stored inthe dark at 4°C until they were viewed.

Rhesus primary dorsal root ganglia culturesChamber slides (two wells) with detachable cultureslides that were previously coated with poly-D lysine(BD Biosciences, Franklin Lakes, NJ, USA) were coatedwith mouse laminin (Invitrogen) at a final concentrationof 10 μg/mL for a minimum of two hours before seedingthe cells. Just before plating the DRG cells, the lamininwas removed and wells were rinsed twice with steriledeionized water. Cells were seeded immediately afterremoval of the laminin. DRG from two adult rhesusmonkeys were obtained at necropsy and transported im-mediately on ice in high glucose (D)MEM F12 (Invitrogen)containing penicillin and streptomycin (P/S) (1 × of10,000 units of penicillin and 10,000 μg/mL of strepto-mycin) (Invitrogen) to the biosafety chamber. The DRGwere transferred to a petri dish containing Hanks balancedsalt solution (HBSS) (Invitrogen). The nerve trunks werecut away using sterile number 21 scalpel blades (PersonnaMedical). The tissue from about four to six DRG fromeach animal was minced and transferred to a tubecontaining 5 mL of 0.25% trypsin ethylenediaminetetraacetic acid (EDTA) (Invitrogen) with 1,000 units ofDNAse (Sigma). The minced tissue was transferred to awater bath set at 37°C for 10 to 15 minutes with intermit-tent shaking. The trypsinization was stopped by adding25 mL of complete (D)MEM medium containing 10% FBS(Fisher Scientific) and P/S. The complete cell suspensionwas centrifuged at 1800 to 2000 rpm (300 to 350 g) forten minutes at room temperature (18°C). The cell pelletwas transferred to a clean tube containing 5 mL ofcomplete (D)MEM F12, supplemented with NGF-7S(Invitrogen) at a concentration of 50 ng/mL. (D)MEM wassupplemented with fresh L-glutamine at a final concentra-tion of 2 mM. The cells were counted using a hemo-cytometer and resuspended to a final count of 2 × 105/mL.Cells were seeded at a count of 1 × 105 cells per well, byseeding 500 μl of the above suspension per well. Chamberswere left in the humidified CO2 incubator for one hour,after which a volume of 1.5 mL of complete (D)MEM F12


medium containing 10% FBS and P/S supplemented withfresh L-glutamine (2 mM), and NGF (50 ng/mL) wasadded gently to each well drop by drop and left to incu-bate. Cultures were maintained for a period of six to sevendays with medium changes every three to four days. TheDRG culture protocol was adapted from previously pub-lished protocols for the isolation of DRG neurons fromembryonic or adult rats, [24].

Human Schwann cell culturesCryopreserved human Schwann cell (HSC) culturesobtained from ScienCell Inc., (Carlsbad, CA, USA), (iso-lated from adult human spinal nerves) were revived intissue culture flasks coated with poly-L-lysine as per themanufacturer’s instructions and maintained in the sup-plied Schwann cell medium (SCM) consisting of basalmedium, 5% FBS, 1% Schwann cell growth supplementand 1 x antibiotic mixture of penicillin/streptomycin.Several aliquots of the above-revived culture (passagetwo) were frozen back as per the manufacturer’s instruc-tions and thawed and revived as needed for experiments.Cultures that were 80% confluent were trypsinized andre-seeded onto poly-L-lysine chamber slides containingtwo wells at a seeding density of 1 × 104 cells per well,as recommended by ScienCell Inc., after which theywere maintained in SCM for three days prior to com-mencing experiments. Extra wells were seeded to evalu-ate final cell count prior to incubation with live B. burgdorferi at the desired multiplicity of infection (MOI).Duplicate wells were seeded for each condition for eachof the two experiments, set up using different passage-two stocks. Both basal media and Schwann cell sup-plement were supplied by the manufacturer and theircomposition is proprietary. HSC maintained on poly-L-lysine-coated chamber slides were used for the eva-luation of phenotypic markers and secreted immunemediators using a Human 23-plex Cytokine-ChemokineArray kit (Millipore) described below.

Immunofluorescence staining and confocal microscopyfor detection of the expression of Schwann cell, neuronaland satellite glial cell markersMedium was removed from HSC cultures or DRG cul-tures and cells were fixed in 2% paraformaldehyde (PFA)followed by post-fixation permeabilization using a mix-ture of ethanol:acetic acid (2:1) (Sigma) for five minutesat −20°C. The slides were then detached from the cham-ber and processed for immunofluorescence staining aspreviously described [25].Schwann cell cultures were evaluated for the expression

of myelin basic protein (MBP), using rabbit polyclonalanti-human MBP Clone AB 980 at 1:100 (Millipore,Billerica, MA, USA), and CD-90 using mouse monoclonalIgG1 anti-human THY-1 (CD90) at 10 μg/mL (Millipore),

in addition to CNPase, p75 NTR and S-100, while DRGcultures were evaluated for the expression of NeuN, GFAP,S-100, CNPase and p75 NTR, as described above in thesection entitled ‘Immunofluorescence staining for detec-tion of intracytoplasmic immune mediators’.Confocal microscopy was performed using a Leica TCS

SP2 confocal microscope equipped with three lasers (LeicaMicrosystems, Exton, PA, USA). Images of individual chan-nels were merged to obtain images containing all channels.Photoshop CS3 (Adobe Systems Inc., San Jose, CA, USA)was used to assign colors to each fluorochrome.

Evaluation of the role of inflammation in mediatingneuronal apoptosis in dorsal root ganglia cultures usingthe anti-inflammatory drug dexamethasoneDRG cell cultures were seeded as described above inchamber slides for evaluation of apoptosis or for evalu-ation of immune mediators and maintained in growthmedium for six days. Prior to stimulation with live B.burgdorferi, DRG cultures were incubated with variousconcentrations of dexamethasone (water soluble), 5 μM,15 μM and 150 μM (Sigma) for 24 hours at 37°C, afterwhich they were washed and then incubated in freshgrowth medium containing the respective concentra-tions of dexamethasone and live B. burgdorferi at a MOIof 10:1 at 37°C for 24 hours and devoid of P/S. Similarconcentrations of dexamethasone as those mentionedabove have been reported to inhibit the production ofCCL2 in mice microglia [26]. The dexamethasone thatwe used in our experiments is supplied as a water-solubleformulation consisting of dexamethasone and a carriersubstance (2-hydroxypropyl)-β-cyclodextrin (HPC). Theeffect of HPC alone, at respective molar concentrationsfound in the concentrations of dexamethasone that weused in our experiments, was done by incubating DRGcultures as described above in the presence and absence ofB. burgdorferi and the above mentioned concentrations ofcarrier alone at 15 μM, 45 μM and 450 μM, respectively.After 24 hours, culture supernatants were collected

and processed for quantification of inflammatory media-tors, and cells were fixed and evaluated for apoptosis bythe in situ terminal deoxynucleotidyl transferase medi-ated UTP nick end labeling (TUNEL) assay as describedbelow. Medium controls that were pretreated and thenincubated with the same respective concentrations ofdexamethasone but without the addition of live B.burgdorferi were also included.

Evaluation of apoptosis by in situ TUNEL assayCells contained in chamber slides were labeled for NeuNby immunofluorescence staining as described above. Slideswere then fixed with 2% PFA, washed three times with PBSby rinsing the slides in PBS and holding them in PBS fortwo minutes between washes. Slides were then subjected


to the TUNEL-ApopTagPlus fluorescein in situ apop-tosis assay (Chemicon, Temecula, CA, USA) as per themanufacturer’s instructions. Slides were then mountedas described above and stored at 4°C in the dark untilviewed. The percentage of apoptotic neurons from tenfields was evaluated from each chamber area by coun-ting the total number of NeuN-positive cells (at least500 cells) from each of the chamber areas, followed bythe percentage of cells that showed co-localization ofboth the TUNEL signal and NeuN expression. Allcounts were made by viewing slides under a fixedmagnification of 63X (corresponding to an area of0.05 mm2) using the confocal microscope. Cultureswere also stained with S-100 prior to doing the TUNELassay as described above to detect apoptotic satelliteglial cells.

Stimulation of human Schwann cell cultures with liveB. burgdorferi for evaluation of immune mediatorsB. burgdorferi strain B31 5A19 passage 3 was prepared asdescribed above. The HSC cultures were washed in SCMdevoid of P/S. The B. burgdorferi culture was resuspendedin SCM devoid of P/S, at the desired MOI. Controls withno spirochetes were also included. Cultures were incu-bated for 48 hours in a humidified 5% CO2 incubator setat 37°C. At the 48-hour time point culture supernatantswere collected for evaluation of inflammatory mediators.Culture supernatants were centrifuged at 4°C at 2000 × gto remove any suspended bacteria and the supernatantwas aliquoted and stored at −70°C until used.

Evaluation of immune mediators from culturesupernatantsThe concentrations of cytokines and chemokines presentin the culture supernatants from rhesus DRG werequantified using the MILLIPLEX MAP Non-HumanPrimate Cytokine Magnetic Bead Panel - Premixed 23Plex, PCYTMG-40 K-PX23 Cytokine-Chemokine Arraykit (Millipore) following the manufacturer’s instructions.The analytes detected by this panel are: G-CSF, GM-CSF,IFN-γ, IL-10, IL-12/23 (p40), IL-13, IL-15, IL-17, IL-18,IL-1ra, IL-1β, IL-2, IL-4, IL-5, IL-6, IL-8, CCL2, CCL3,CCL4, TGF-α, TNF-α, VEGF and sCD40L. The concentra-tions of cytokines and chemokines present in the culturesupernatants from HSC cultures described above werequantified using the MILLIPLEX Human Cytokine/Chemokine - Premixed 14 Plex, MPXHCYTO60KPMX14 Cytokine-Chemokine Array kit (Millipore) fol-lowing the manufacturer’s instructions. The analytesdetected by this panel are: GM-CSF, IFN-γ, IL-10, IL-12(p70), IL-13, IL-1β, IL-2, IL-4, IL-5, IL-6, IL-7, IL-8, CCL2and TNF-α. The multiplex plate was read using a Bio-Plex200 Suspension Array Luminex System (Bio-Rad, Hercules,CA, USA).

Statistical evaluationThe unpaired-two tailed t test was used to evaluate thestatistical significance between means of data sets, usingGraphpad Prizm software (Graph Pad Software Inc.) ver-sion 4. A P value of 0.05 or lower was considered to bestatistically significant.

ResultsVisualization of CCL2, IL-6 and IL-8 in cells from dorsalroot ganglia tissue explants after ex vivo stimulation withlive spirochetesThe DRG tissue explants stimulated ex vivo with spiro-chetes in the presence of brefeldin A showed localizationof CCL-2 in sensory neurons (Figure 1A). CCL-2, whichis stained green in this tissue, is localized inside the neu-rons giving a yellow signal due to the co-localization ofred and green within the neurons. CCL-2 is also seen inareas occupied by satellite glial cells and dorsal root inthe DRG. CCL-2 in green is seen co-localizing with thesatellite glial cell marker GFAP (Figure 1B). Both satelliteglial cells and sensory neurons in the DRG that areknown to stain positive for S-100 [27], seen in red, showthe presence of CCL-2 (Figure 1C). CCL-2 is also seento co-localize with Schwann cells in the dorsal root,staining positive with p75NTR, seen in red (Figure 1D),while remaining unstained nerve tissue is seen as gray.The spirochetes appear blue in Figure 1A-1D.Similarly, the cytokine IL-6 was seen to be localized in

sensory neurons (Figure 1E and Figure 1F, respectively)and in satellite glial cells (Figure 1F). Schwann cells alsoshowed the presence of IL-6 (Figure 1G).The chemokine IL-8 was localized inside satellite glial

cells (Figure 1H) and in Schwann cells (Figure 1I) butnot in neurons. The lack of IL-8 production in neuronswas confirmed by staining the section for detection ofIL-8 and with NeuN, a specific marker for neurons (notshown). CCL2, IL-6 and IL-8 were not detected in con-trol sections of DRG explants that were held in mediumcontaining brefeldin A for four hours in the absence ofB. burgdorferi. Representative images showing the ab-sence of CCL-2, IL-6 and IL-8 (green signal) in controlsections (no B. burgdorferi) that were also stained in par-allel for S-100, CNPase or NeuN (all appearing red) areshown in Figure 1J, Figure 1K, and Figure 1L, respect-ively. The unstained tissue appears gray. No signal wasdetected in sections stained with isotype controls for re-spective primary antibodies followed by correspondingsecondary antibodies for the mediators or the cellmarkers (not shown).

Phenotypic cell markers expressed in primary cultures ofdorsal root ganglia cellsIn order to permit a more detailed evaluation of the abil-ity of B. burgdorferi spirochetes to elicit inflammation

Figure 1 Visualization of CCL2, IL-6, IL-8 and B. burgdorferi. (A) CCL-2 (green) inside neurons stained red with NeuN. The signal for CCL-2 isalso in satellite glia and dorsal root nerves. (B) CCL-2 in green in satellite glia stained red with GFAP-cy3. (C) CCL-2 in green in satellite glial andneurons stained red with S-100. CCL-2 also co-localizes with Schwann cells staining positive with p75NTR, in red in the dorsal root (D). Unstainednerve tissue is gray as imaged by differential interference contrast (DIC). The spirochetes in A-D appear blue stained with antibody against wholeB. burgdorferi, in combination with a Zenon labeling Kit Alexa 647. (E) IL-6 (green) localized inside neurons staining red with NeuN. The signal forIL-6 is also in areas occupied by satellite glia. (F) IL-6 in green within the satellite glia and neurons stained red by S-100. The signal for IL-6 is alsoseen in areas occupied by sensory neurons. (G) IL-6 (green) in Schwann cells stained with p75NTR, in red in the dorsal root; the remainingunstained nerve tissue is gray as imaged by DIC. The spirochetes appear blue in E-G. (H) IL-8 (green) inside satellite glia stained positive for GFAP(red). The signal for IL-8 is not seen in areas occupied by neurons. (I) IL-8 (green) co-localized within Schwann cells stained with CNPase (red) inthe dorsal root. Spirochetes appear blue in H and I. (J, K and L) show the absence of CCL2 , IL-6 and IL-8, respectively, in control sections (greensignal) that were also stained in parallel for S-100, CNPase or NeuN (all appearing red). The unstained tissue appears gray as imaged by DIC.Results indicate representative images of DRG explants from three adult rhesus macaques. CNPase, 2′,3′-cyclic nucleotide 3′-phosphodiesterase;DRG, dorsal root ganglia; GFAP, glial fibrillary acid protein.



and apoptosis in DRG cells, we set up in vitro culturesof these cells and characterized their phenotypes. Theprimary rhesus DRG cultures consisted primarily of amat of sensory neurons that stained positive for theneuronal marker NeuN and a few stray satellite glialcells that stained positive for the glial cell marker S-100(Figure 2A). The satellite glial cells also showed the ex-pression of GFAP (Figure 2B). The sensory neurons inthe DRG cultures did not stain positive for S-100, con-trary to what we had observed in sensory neurons withinDRG tissue explants (Figure 1C and Figure 1F). The nu-clei of all cells in Figure 2A and 2B appeared blue due tostaining with the nuclear stain TOPRO3. DRG cultureslides that were incubated with respective isotype con-trols of primary antibodies and corresponding secondaryantibodies did not show any detectable signal (notshown). DRG cultures did not show specific stainingwith antibodies to the Schwann cell markers CNPase orp75NTR.

Expression of MBP, CNPase and p75NTR in humanSchwann cell culturesAs we were not able to detect the presence of Schwanncells in the DRG cultures described above, we resorted tocultivating commercially available HSCs and confirmedtheir phenotype and purity. HSC cultures showed specificstaining with MBP and did not show the expression ofCD-90 (Figure 3A). Our HSC cultures also showed the ex-pression of the myelinating marker CNPase and theSchwann cell marker p75NTR (Figure 3B). The HSC cul-tures did not show any signal when stained with isotypecontrols for the various primary antibodies at the relevantconcentrations followed by secondary antibodies. A

Figure 2 Expression of the neuronal marker NeuN and satellite glial cPrimary rhesus DRG cultures showing a mat of sensory neurons staining poglial cells staining positive for the glial cell markers S-100 and GFAP (B). Thnuclear stain TOPRO3. DRG, dorsal root ganglia; GFAP, glial fibrillary acid pr

representative image of HSC cultures stained with isotypecontrol for mouse IgG1 followed by goat anti-mouseAlexa Fluor 568 is shown in Figure 3C. The nuclei of cellsappear blue in Figure 3A, 3B and 3C due to staining withthe nuclear stain TOPRO3. The HSC expressed themarkers known to be present on adult HSCs, such asMBP [28], CNPase [29] and p75 NTR [30] but did notshow the expression of CD90/THY-1, a phenotypicmarker of perineural fibroblasts [31], thus signifying thepurity of the HSC culture.

Effect of dexamethasone on the pro-inflammatoryresponse elicited by B. burgdorferi in primary rhesusdorsal root ganglia culturesLive B. burgdorferi spirochetes incubated with rhesusDRG cultures for 24 hours at a MOI of 10:1 inducedsignificantly elevated levels of CCL2 (Figure 4A), IL-6(Figure 4B) and IL-8 (Figure 4C) compared to the levelsinduced in medium controls. The concentration ofCCL2 surpassed 20,000 pg/mL, whereas the constitutivelevel of this chemokine that was produced in mediumalone was around 8,000 pg/mL (Figure 4A). The basalconcentration of IL-6 was 4,000 pg/mL but reached al-most 8,000 pg/mL (Figure 4B) and the basal level of IL-8production in DRG cultures was around 800 pg/mL butreached about 5,000 pg/mL when stimulated with liveB. burgdorferi for 24 hours (Figure 4C).Dexamethasone significantly reduced the levels of CCL2

(P <0.01), IL-6 (P <0.05) and IL-8 (P <0.05) as induced bylive B. burgdorferi (MOI of 10:1) in DRG cultures after24 hours in a dose dependent manner as shown inFigures 4A, 4B and 4C, respectively. We confirmed that theanti-inflammatory effect of the dexamethasone formulation

ell markers GFAP and S-100 in primary rhesus DRG cultures.sitive for the neuronal marker NeuN (red) (A) and a few stray satellitee nuclei of all cells in A and B appear blue due to staining with theotein.

Figure 3 Expression of MBP, CNPase and p75NTR in HSC cultures. (A) shows HSC cultures staining with MBP (red) but not CD-90. (B) showsthe expression of the myelinating marker CNPase (red) and the Schwann cell marker p75NTR (green) in HSC cultures. A representative image ofHSC cultures stained with isotype control for mouse IgG1 followed by goat anti-mouse Alexa Fluor 568 is shown in (C). The nuclei of cells appearblue in A, B and C due to staining with nuclear stain TOPRO3. CNPase, 2′,3′-cyclic nucleotide 3′-phosphodiesterase; HSC, human Schwann cell;MBP, myelin basic protein.


was due to its dexamethasone fraction and not due to thecarrier substance (HPC), as HPC alone at 15 μM, 45 μMand 450 μM present in the dexamethasone solutions at theconcentrations used above was unable to reduce the levelsof B. burgdorferi-induced immune mediators (Figure 4A,4B and 4C, respectively). Data represent mean values andstandard deviations between values of two independentexperiments.

Inhibiting effect of dexamethasone on apoptosis inducedby B. burgdorferi in primary rhesus dorsal root gangliaculturesLive B. burgdorferi induced elevated levels of apoptosisof sensory neurons, as detected by the in situ TUNELassay in primary rhesus DRG cultures, after 24 hours ofincubation. Apoptosis visualized by confocal microscopyin cells that were held in medium alone is shown inFigure 5A, after incubation with live B. burgdorferi atMOI of 10:1 in Figure 5B and with B. burgdorferi plusdexamethasone at 15 μM in Figure 5C, respectively. Thesensory neurons appear red due to staining with theneuronal marker NeuN. The TUNEL signal can be visu-alized in green while the nuclei of all cells appear blue dueto staining with the nuclear stain TOPRO3. Figure 5Dshows percent apoptosis as measured by the in situTUNEL assay after 24 hours of incubation of DRG cul-tures in medium (2.41% ± 0.62), B. burgdorferi alone at aMOI of 10:1 (13.82% ± 1.39) and B. burgdorferi + dexa-methasone at 5 μM, 15 μM and 150 μM in medium.Dexamethasone induced a significant reduction in B.burgdorferi-dependent apoptosis at 15 μM (7.39 ± 0.74)and 150 μM (5.32 ± 0.84) (P <0.05), respectively. Themean percent apoptosis and standard deviations quanti-fied from ten microscope fields (a total of 500 cells) foreach condition is shown in Figure 5D. The results repre-sent the mean and standard deviation of values obtainedfrom two independent experiments. No apoptosis was

seen in the satellite glial cells in the primary rhesus DRGcultures, (not shown).

Pro-inflammatory response induced by B. burgdorferi inHSC culturesAs we had detected the presence of CCL2, IL-6 and IL-8 in the dorsal roots of the DRG explants stimulatedex vivo with live B. burgdorferi, we evaluated thepotential of live spirochetes to induce inflammatorymediators in in vitro cultures of pure HSC. Live B.burgdorferi spirochetes incubated with HSC culturesfor 48 hours at a MOI of 10:1 and 50:1 induced signifi-cantly elevated levels of CCL2 (Figure 6A), IL-6(Figure 6B) and IL-8 (Figure 6C) as compared to thelevels induced in medium controls. The concentrationof CCL2 surpassed 21,000 pg/mL and 32,000 pg/mL ata MOI of 10:1 and 50:1, respectively, whereas the con-stitutive level of this chemokine was about 13,000 pg/mL (Figure 6A). The basal concentration of IL-6 wasonly about 300 pg/mL but reached more than 1,150 pg/mL and 2,500 pg/mL at a MOI of 10:1 and 50:1, re-spectively (Figure 6B). Similarly, the basal concentra-tion of IL-8 that was around 6,400 pg/mL reachedmore than 16,000 pg/mL and 23,000 pg/mL at a MOIof 10:1 and 50:1, respectively (Figure 6C). Data repre-sent mean values and standard deviations betweenvalues of two independent experiments.

DiscussionThe pathogenesis of Lyme disease neuropathies is poorlyunderstood. B. burgdorferi infection may damage neuralcells by the direct action of spirochetes or spirochetalproducts on glial and neuronal cells. It is also possiblethat spirochetes induce cytotoxic or inflammatory medi-ators locally in glial, neuronal or endothelial cells and,thus, cause indirect damage. Infiltrating immune cellsand/or the presence of cross-reactive antibodies to self-

Figure 4 Effect of dexamethasone on the pro-inflammatoryresponse elicited by B. burgdorferi in primary rhesus DRGcultures. The levels of CCL2 (A), IL-6 (B) and IL-8 (C), respectively, inrhesus DRG cultures stimulated with live B. burgdorferi spirochetesfor 24 hours at a MOI of 10:1 and in medium controls (* P <0.05,** P <0.01) in the presence and absence of dexamethasone andcarrier HPC are shown. Data represent mean values and standarddeviations between values of duplicate wells that were seeded fromDRG cells that were isolated from two adult rhesus macaques. DRG,dorsal root ganglia; HPC, (2-hydroxypropyl)-β-cyclodextrin; MOI,multiplicity of infection.


antigens at the site of infection/inflammation may alsobe deleterious to neural cells.Neuronal proteins, anti-myelin antibodies and cells se-

creting antibodies to MBP have been detected in the CSF

of patients with LNB, indicating possible glial and neur-onal damage [32,33]. The antigenic determinants on the41 kDa flagellar protein of B. burgdorferi are shared byseveral human tissue components such as Schwann cellsfrom the PNS [34]. Long-term murine intrathecal expos-ure to a lipoprotein of B. burgdorferi, outer-surface proteinC, resulted in axonal damage. Intrathecal exposure to B.burgdorferi lipoproteins may be one of the causes of theneurologic manifestations of Lyme disease [35]. The in-volvement of sensory ganglia in human LNB has also beenpreviously documented [15,36]. One rare peripheral ner-vous system manifestation of LNB is the Guillain-Barré-like syndrome, a prototype of immune-mediated periph-eral neuropathies where neither the initial event nor theantigen that triggers the immune reaction is known. Auto-immune mechanisms similar to those suggested in MShave also been implied in the pathogenesis of central ner-vous system LNB [37].Our earlier findings in the rhesus model of peripheral

LNB of both the early disseminated and chronic phases inthe PNS mirror several aspects of these forms of the dis-ease in humans [38,39]. The primary findings of axonaldegeneration and regeneration, and multifocal nerve le-sions showing perivascular inflammatory cellular infil-trates have been documented in almost all patients withLyme-associated peripheral neuropathy [15,16,40-42]. Theresults of these studies suggest that immune mediatedneuronal and glial cell damage could be involved in theneuropathy of LNB.Cytokines and chemokines are key immune mediators

that play an important role in promoting CNS injury invarious kinds of inflammatory neurodegenerative diseases[43-47]. Importantly, various inflammatory cytokines andchemokines have also been reported in the CSF of patientswith LNB [48-51]. The potential of B. burgdorferi to in-duce cytokines, chemokines and other inflammatorymediators in glial and neuronal cells as well as glialand neuronal apoptosis has been well documented[20,23,25,52-56].In this study we explored the potential of the Lyme

disease bacteria to cause inflammation in tissue explantsof rhesus DRG, as well as in primary cultures of rhesusDRG cells and human Schwann cells, as a representationof the Schwann cells that ensheath the dorsal roots. Pri-mary cultures of DRG cells from rhesus monkeys mayprove useful in understanding the mechanisms involvedin Lyme peripheral neuropathy, as well as in other hu-man peripheral neuropathies [57].We documented the ability of B. burgdorferi to elicit

the production of IL-6, IL-8, and CCL2 in cells of theDRG and to induce the death of sensory neurons inDRG cell cultures. The ability of sensory neurons of theDRG to express CCL2 has also been documented in painmodels [58]. Our in vitro DRG culture did not favor the

Figure 5 Protective effect of dexamethasone on apoptosis induced by B. burgdorferi in primary rhesus DRG cultures. (A-C) showrepresentative images of sensory neurons in DRG cultures (red) staining with the neuronal marker NeuN, exposed to medium (A), live B.burgdorferi at a MOI of 10:1 (B) and B. burgdorferi plus dexamethasone 15 μM (C), respectively after 24 hours of incubation. Apoptotic nuclei arevisualized in green as detected by the in situ TUNEL assay. The nuclei of all cells appear blue due to staining with the nuclear stain TOPRO3.(D) shows percent apoptosis as measured by the in situ TUNEL assay and quantified by confocal microscopy after 24 hours of incubation of DRGcultures exposed to medium or B. burgdorferi at a MOI of 10:1 in the presence and absence of dexamethasone (* P <0.05). Data represent percentapoptosis mean values from DRG cells that were isolated from two adult rhesus macaques. Values for each animal were determined in duplicate.Error bars represent standard deviations. DRG, dorsal root ganglia; MOI, multiplicity of infection; TUNEL, terminal deoxynucleotidyl transferasemediated UTP nick end labeling.


growth of Schwann cells, which are known to ensheaththe dorsal root axons in the DRG, possibly because theculture medium was tailored to supporting the neurons.Interestingly, the few satellite glial cells that were presentin our DRG cultures did not undergo apoptosis in re-sponse to B. burgdorferi. We had previously observedthe apoptosis of satellite glial cells in DRG of rhesusmonkeys infected intrathecally with live B. burgdorferi[20]. It is possible that there are additional regulatoryfactors that come into play in the in vivo environmentthat are absent in the in vitro culture system. Specific-ally, this difference may have been brought about by theabsence of Schwann cells in the DRG cultures, especially

considering their documented contribution to inflamma-tion in response to B. burgdorferi.Using rhesus DRG tissue explants we identified the

phenotypes of the producer cells as satellite glial cells bythe expression of glial markers such as S-100 [59,60]and GFAP. Like other glial cells, the satellite glial cellsare known to respond to nerve injury by up-regulatingGFAP [61]. We identified Schwann cells in rhesus DRGtissue explants by the expression of p75NTR andCNPase [29,30,62]. These cells, in addition to DRG sat-ellite cells, produced IL-6, IL-8 and CCL2, while neu-rons, which were characterized by the expression ofNeuN [63], produced IL-6 and CCL2 but not IL-8. The

Figure 6 Pro-inflammatory response induced by B. burgdorferiin HSC cultures. The levels of immune mediators CCL2 (A), IL-6 (B)and IL-8 (C) in HSC cultures exposed to live B. burgdorferispirochetes for 48 hours at a MOI of 10:1 and 50:1, respectively, andin medium controls are shown (* P <0.05, ** P <0.01). Data representmean values and standard deviations between values of duplicatewells that were seeded for each condition for each of the twoexperiments set up using separate passage stocks. HSC, humanSchwann cells; MOI, multiplicity of infection.


DRG cell cultures as well as the cultures of HSC stimu-lated with live B. burgdorferi produced IL-6, IL-8 andCCL2. Since we did not find IL-8 to be produced byneurons in rhesus DRG explants, it is likely that satelliteglial cells contributed the IL-8 found in the DRG cellculture supernatants.These results support our hypothesis and show that

innate responses of neuronal and glial cells of the DRGto B. burgdorferi mediate inflammation and that neur-onal apoptosis occurs in this context. In agreement with

this notion we found that the anti-inflammatory drugdexamethasone reduced both the levels of inflammatorymediators and neuronal apoptosis as induced by B.burgdorferi, suggesting that the two phenomena may becausally related.Cytokine/chemokine signaling and apoptosis are of

key importance in the regulation of neuroinflammatoryresponses [43-47]. Since DRG axons project centrallyinto the spinal cord and peripherally into the spinalnerves, inflammation and cell death in the DRG elicitedby the Lyme disease spirochete could affect neuronalsurvival and function both in the CNS and PNS. Further,as the sensory neurons of the DRG play a key role in thesensation of pain, inflammation in glial and neuronalcells and cell death in the DRG could also modulate thepain response [64]. Neurogenic pain secondary to radi-culitis or inflammation of the dorsal roots is often theearliest and sometimes only symptom in patients withLNB [65]. It typically radiates from the spine into the ex-tremities or trunk, and is described as ‘sharp or jabbing’pain [16]. The immune mediators IL-6, IL-8 and CCL2that we found to be elevated in the DRG cultures ex-posed to live B. burgdorferi have been reported to play arole in modulating inflammation and the pain response[66-69]. IL-8 is known to induce expression of matrixmetalloproteinases, cell cycle and pro-apoptotic proteins,and cell death in neurons [70]. IL-6 and CCL2 have beenreported to increase the sensitivity of sensory neurons topain [71-73].The expression of the chemokine CCL2 and its recep-

tor (CCR2) is also up-regulated by DRG neurons in ro-dent models of neuropathic pain [74]. Disruption ofCCL2 signaling has been shown to block the develop-ment of neuropathic pain [75]. CCL2 is also known tobe involved in the signaling and upregulation of severalgenes and proteins that participate in the signal trans-duction of the pain response both in the DRG and in thespinal cord [72,73]. This chemokine also functions as aneuromodulator in DRG neurons [74]. We observed thelevels of CCL2 to be the highest among the immune me-diators elicited by B. burgdorferi in both DRG cell andHSC cultures. Similarly, we have reported high levels ofCCL2 in the CSF of rhesus macaques infected with B.burgdorferi [20]. It is possible that CCL2 is a majorplayer in orchestrating inflammation as well as the painresponse in LNB.The local application of IL-6 to the DRG of rats has been

shown to induce TNF-α and results in apoptosis of DRGcells [76]. Earlier, we also reported the presence of IL-6 inthe sensory neurons of the DRG in rhesus macaques thatwere inoculated intrathecally with B. burgdorferi [20].Patients with LNB sometimes have persistent symptomssuch as fatigue, cognitive difficulties, depression and paineven after appropriate antibiotic treatment. Because of the


subjective nature of these complaints, many of these pa-tients are believed to have a primary psychiatric diag-nosis, such as depression or somatization disorder.However, experiments such as those presented hereraise the possibility that some of these complaints maybe associated with inflammatory biochemical changesin the CNS. Elevated levels of IL-6 can cause symptomsof fatigue and malaise, common to many infectiousconditions, as well as Lyme disease [77]. Research inother animal models of peripheral neuropathy hasdemonstrated that peripheral inflammation alone cantrigger the brain cytokine system via afferent neuralpathways from the periphery to the CNS [78]. Similarcascades of inflammation and apoptosis could also beinvolved in the DRG in LNB. Ongoing cytokine activa-tion in the nervous system could contribute to thepersistent symptoms of fatigue, pain and cognitive dys-function that patients sometimes continue to experi-ence despite having been treated for Lyme disease.It is possible that B. burgdorferi, as well as the media-

tors elicited in cells of the DRG and Schwann cells thatwe report here, could contribute to mediating inflamma-tory and apoptotic signaling cascades in Schwann cells,the myelinating cells of the PNS. This, in turn, may re-sult in axonal degeneration [79].The potential of Schwann cells to initiate the process

of Wallerian degeneration by releasing pro-inflammatorycytokines that are involved in leukocyte recruitment anddifferentiation (for example, IL-1β, CCL2, IL-8 and IL-6)has been documented [80]. As we found the inflamma-tory mediators IL-6, IL-8 and CCL2 in our HSC culturesupernatants stimulated with live B. burgdorferi, as wellas in the Schwann cells in the dorsal roots of the DRGtissue explants incubated with live B. burgdorferi, it ispossible that similar mechanisms of inflammatory medi-ated axonal damage could be contributing to the periph-eral neuritis seen in LNB.The high levels of CCL2 that we found to be elicited in

DRG neurons and satellite glial cells, as well as in Schwanncells in response to B. burgdorferi, could trigger mecha-nisms of demyelination in the PNS similar to those thoughtto cause CNS demyelination in MS and experimental auto-immune encephalomyelitis (EAE) [81,82]. CCR2, the recep-tor of CCL2, has been documented to be involved inmacrophage recruitment to the injured PNS [83].The ability of B. burgdorferi to activate the innate de-

fense mechanisms of the host [84] and particularly in cellsof the CNS is well documented [48,51,85-88]. It is possiblethat similar signaling cascades could be involved in trigger-ing the immune mechanisms that result in the pathogen-esis of peripheral LNB. We propose that inflammation ofnerve roots and DRG and subsequent apoptosis in theDRG could be early events that contribute to peripheralneuropathy in Lyme neuroborreliosis.

ConclusionsIn this model, B. burgdorferi induced an inflammatoryresponse and neuronal apoptosis of DRG. These patho-physiological processes could contribute to peripheralneuropathy in LNB.

AbbreviationsCCL2: Chemokine (C-C) motif ligand 2; CCR2: C-C chemokine receptor 2;CD90: Cluster of differentiation 90; CNPase: 2′, 3′- cyclic nucleotide 3′-phosphodiesterase; CNS: Central nervous system; CSF: Cerebrospinal fluid;DIC: Differential interference contrast; DRG: Dorsal root ganglia; FBS: Fetalbovine serum; FSG: Fish skin gelatin; G-CSF: Granulocyte colony stimulatingfactor; GFAP: Glial fibrillary acidic protein; GM-CSF: Granulocyte macrophagecolony stimulating factor; HPC: 2- hydroxypropyl β- cyclodextrin; HSC: HumanSchwann cells; IFN-γ: Interferon gamma; IL-1ra: Interleukin 1 receptorantagonist; IL: Interleukin; LNB: Lyme neuroborreliosis; MBP: Myelin basicprotein; MCP: Monocyte chemotactic protein; MOI: Multiplicity of infection;MS: Multiple sclerosis; NeuN: Nneuronal nuclear protein N; NGF: Nervegrowth factor; NGS: Normal goat serum; P/S: Penicillin and streptomycin;p75NTR: Neurotrophin receptor p75; PBS-FT: Phosphate buffered saline fishskin gelatin Triton-X-100; PBS: Phosphate buffered saline;PFA: Paraformaldehyde; PNS: Peripheral nervous system; sCD40L: Solublecluster of differentiation 40 ligand; SCM: Schwann cell medium; TGF-α: Transforming growth factor alpha; TNF-α: Tumor necrosis factor alpha;TUNEL: Terminal deoxynucleotidyl transferase mediated UTP nick endlabeling; VEGF: Vascular endothelial growth factor.

Competing interestsThe authors declare that they have no competing interests.

Authors’ contributionsGR participated in the design of the experiments, conducted cell culture ofDRG and HSC, ex vivo DRG tissue explant experiments, multiplex ELISA dataanalysis, immunofluorescence staining and confocal microscopy and draftedthe manuscript. LSG helped in cell culture, immunofluorescence staining andmultiplex ELISA data analysis of HSC. FI helped in establishing primary DRGcell cultures. JDE contributed to editing the manuscript. MTP conceived ofthe study, contributed to the design of the experiments, and to drafting andediting the manuscript. All authors have read and approved the final versionof the manuscript.

AcknowledgementsThis project was supported by the National Institute of Neurologic Disordersand Stroke through grant NS048952, and by the National Center forResearch Resources and the Office of Research Infrastructure Programs (ORIP)of the National Institutes of Health through grant P51OD011104/P51RR000164. We thank the TNPRC Pathogen Detection and QuantificationCore Laboratory for help with the multiplex ELISA assays. Robin Rodriguezfrom the TNPRC Media Laboratory is gratefully acknowledged for assistingwith formatting the figures.

Author details1Division of Bacteriology and Parasitology, Tulane National Primate ResearchCenter, Covington, LA, USA. 2Department of Cell and Molecular Biology,Tulane University, New Orleans, LA, USA. 3Department of Neurology,Louisiana State University Health Sciences Center, New Orleans, LA, USA.

Received: 9 April 2013 Accepted: 1 July 2013Published: 18 July 2013

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doi:10.1186/1742-2094-10-88Cite this article as: Ramesh et al.: The Lyme disease spirochete Borreliaburgdorferi induces inflammation and apoptosis in cells from dorsal rootganglia. Journal of Neuroinflammation 2013 10:88.

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